We investigated the role of the L3T4 molecule in mitogen and antigen-initiated signal transduction in the L3T4(+) murine T cell hybridoma, 3DT52.5.9 and an L3T4(-) variant, 3DT52.5.24. Both Concanavalin A (Con A) and specific antigen stimulated increases in cytosolic-free calcium ([Ca2+]i), phosphatidylinositol turnover, and interleukin-2 (IL-2) production in both cell lines. About 85% of the stimulated rise in [Ca2+]i was from an extracellular source. Anti-L3T4 monoclonal antibody (MAb) inhibited 90% of antigen- and 50% of Con A-stimulated increases in [Ca2+]i and IL-2 production but had no effect on the ability of either activation signal to stimulate phosphatidylinositol turnover in the parent L3T4(+) cells. Stimulus-response coupling in the L3T4(-) cells was unaffected by the MAb. The anti-L3T4-insensitive increase in [Ca2+]i induced by Con A was inhibited by EGTA, suggesting that this mitogen also stimulated an influx of Ca2+ via an additional transport mechanism distinct from that stimulated by antigen. The fact that anti-L3T4 antibodies inhibit antigen and Con A-stimulated Ca2+ transport and IL-2 production without affecting phosphatidylinositol turnover suggests that L3T4 may play a critical role in modulating the activation of the T cell receptor-associated Ca2+ transporter in T cell stimulus-response coupling.